人骨髓来源问充质干细胞对新生大鼠高氧肺损伤干预作用的研究

Influence of human bone marrow-derived mesenchymal stem cells on the lung of newborn rats damaged by hyperoxia

目的研究人骨髓来源间充质干细胞对新生大鼠高氧肺损伤的干预作用。方法采用贴壁选择法分离、培养、扩增hMSCs，并予BrdU进行标记；32只3日龄SD大鼠随机分为A、B、C、D4组，每组8只；A组：高氧暴露＋hMSCs注射组，B组：空气暴露＋hMSCs注射组，C组：高氧暴露对照组，D组：空气暴露对照组。A、C组：高氧（95％）暴露后7d，腹腔分别注射含5×10^5MSC的磷酸盐缓冲液（PBS）、单纯的PBS50μl，B、D组：空气暴露后7d，腹腔分别注射含5×10^5hMSCs的PBS、单纯的PBS50μl。注射后3d处死全部动物取肺组织，ELASA法检测肺组织匀浆TNFα、TGFβ1水平；免疫组织化学方法检查肺组织BrdU积分情况，HE染色观察肺组织学形态学结构，做辐射状肺泡计数（RAC）；RT—PCR方法检测各组肺组织Alu序列表达情况。结果（1）A、B、C、D4组TNFα水平分别为142．933±24．017，79．033±11．573，224．088±41．915，76．500±10．373（F=59．970．P=0．000）；而TGFβ1水平分别为1726．484±91．086，1530．359±173．441，2047．717±152．057，1515．777±131．049（F=24．977，P=0．000）。（2）RAC值分别为11．145±1．331，13．941±0．985，9．595±0．672，14．819±1．080（F=43．234，P=0．000）。（3）RT—PCR检测显示A、B组均有Alu序列表达，而C、D组均未见Alu序列表达。免疫组织化学显示A、B组均有BrdU阳性染色细胞，BrdU积分分别为0．230±0．026，0．190±0．015，t=3．769，P=0．002；C、D组均未见阳性染色细胞。结论新生大鼠腹腔注射hMSC后肺组织有hMSCs定植，且与暴露的条件相关；hMSCs可改善新生大鼠因高氧引致的肺组织损伤。

Objective To evaluate whether human mesenchymal stem cells （hMSCs） administration alter the clinical course of hyperoxia-induced lung injury. Methods hMSCs were obtained from bone marrow aspirates from healthy donors after informed consent was signed, hMSCs were separated, cultured, amplified, identified and labeled with BrdU. For BrdU labeling, a sterile stock solution was added to the culture medium 48 h before the end of culture, at a final concentration of 10 μmol/L. Thirty-two 3- day old SD rats from four litters were randomly divided into four groups, as hyperoxia exposed ＋ hMSC group （A）, air-exposed ＋ hMSC group （B）, hyperoxia exposed group （C）, and air-exposed group （D）. The rats from the group A and the group C were placed in a sealed Plexiglas chamber with a minimal in- and outflow, providing six to seven exchanges per hour of the chamber volume and maintaining O2 levels above 95% , while the rats in the group B and the group D were only exposed to room air. Seven days later, all of them were taken out of the chamber, rats in the group A and B were injected intraperitoneally with hMSCs （ 1 × 10^5 in 50 μl of PBS） immediately, while the rats in the group C and D were only treated with 50 μl of PBS 3 days later. All the animals were sacrificed by an injection of sodium pentobarbital （ 120 mg/kg）, perfused with cold 0. 9% NaCl, and the left lungs were removed, the upper lobes of which were ground as tissue homogenates and used for ELISA, while the inferior lobes were stored at -70℃ until use for RT-PCR. The right lungs were fixed in situ for 2 h by the intratraeheal instillation with 10% neutral formalin and then postfixed for 24 h. Sagittal sections （4-μm） of paraffin-embedded middle lobe and upper lobe of the right lung were used for immunohistochemistry and histology, respectively. Results （ 1 ） There was a significant difference in the value of RAC （ raditive alveoli coant） among the 4 groups （ 11. 145 ± 1. 331, 13. 941±0. 985, 9. 595±0. 672,14. 819±1. 080,F =43. 234,P =0. 000）. RAC in group A and C were significantly reduced compared with subjects in group D （ P 〈 0. 05, P 〈 0. 05 ） ; and there was also a significant difference between group A and group C （P 〈 0. 05 ）, but not between group B and D subjects （P〉0.05）. （2） There were significant differences in the levels of both TNFα and TGFβ1 in the homogenate of lungs among the 4 groups （ 142. 933 ± 24. 017, 79. 033 ± 11. 573, 224. 088 ± 41. 915, 76.500±10.373, F=59.970, P =0.000; 1726.484±91.086, 1530.359 ± 173.441, 2047.717 ± 152. 057, 1515. 777 ± 131. 049, F =24. 977 ,P =0. 000）. The levels of TNFα and TGFβ1 were significantly elevated in both group A and group C when compared with subjects in group D （ P 〈 0. 05 for both ）. Concentrations of TNFα and TGFβ1 were both significantly decreased in group A versus group C （ P 〈 0. 05 for both）. There was no significant difference between group B and D subjects in the fields of TNFα and TGFβ1 （P 〉0. 05 for both）. （3） BrdU-labelled cells were observed at alveolar wall and bronehioles in both group A and group B, and there was a significant difference in BrdU-labeled cells between two groups （0. 230±0. 026, 0. 190 ± 0. 015; t = 3. 769, P = 0. 002）, but none was found in group C and group D. Eleetrophoresis of the PCR products showed a 224 bp band, specific for Alu mRNA, in 7 of 8 rats of group A and 5 of 8 rats of group B, respectively, but no such band was found in group C and group D. Conclusion hMSCs administered by intraperitoneal injection could be implanted in the lungs of newborn rats, and they could effectively protect the rats against damage to the lungs caused by hyperoxia.